Applicant's Abstract Diabetes mellitus (D) leads to diabetic cardiomyopathy (DCM) and congestive heart failure. The molecular basis of DCM is only incompletely explored. It is their hypothesis that in DCM hyperglycemia significantly contributes to abnormalities in calcium handling and contractile function and that contractile function in DCM can be improved by expression of calcium handling proteins and changes in the activity of enzymes in the hexosamine and glycosylation pathways. In Aim I they will determine the molecular basis of DM induced abnormalities in calcium handling and explore mechanisms leading to decreased levels of the calcium ATPase of the sarcoplasmic reticulum (SERCA2) and changes in the ryanodine receptors (RyR2). We will also determine sarcoplasmic reticulum (SR) calcium loading, calcium spark patterns and NA+/Ca exchanger activity and the phospholamban (Plb) phosphorylation status. In Aim II they will explore if abnormal calcium handling of the DM heart occurs largely due to exposure to the diabetic milieu or is also contributed to by a lack of insulin signaling at the cardiac myocyte. The preliminary results show that exposing myocytes to hyperglycemia in cell culture and increased hexosamine pathway activity in vivo as it occurs in glutamine fructose 6P amidotransferase (GFAT) transgenic mice and diminishes SERCA2 and RyR2 gene expression. This will be further explored by altering the activity of key enzymes in the hexosamine and glycosylation pathways using adenoviral vectors expressing the appropriate transgene and transgene mice. To assess influences of insulin signaling and changes in the diabetic milieu the applicants are constructing cell type specific insulin receptor null mutant mice using the Cre lox P system. In Aim III they will further explore the preliminary findings that increased SERCA2 expression ameliorates DCM using SERCA2 transgenic mice and, adenoviral vector based delivery of a Plb antibody transgene to rats in vivo. Similar approaches will be used to inhibit enzymes in the hexosamine and glycosylation pathway to improve calcium handling and contractile performance in the presence of persistent hyperglycemia. Novel insights into the molecular basis of calcium flux abnormalities in DCM and new approaches to increase cardiac function will result from this proposal.